【作者】 何艳新；

【导师】 刘超；

【作者基本信息】 郑州大学 ， 外科学， 2012， 硕士

【摘要】 研究背景肝细胞癌在我国是常见的恶性肿瘤之一,是原发性肝癌的主要类型之一。然而其发病机制尚未完全阐明,肝癌的发生发展是一个涉及多基因的变化、多因素、多步骤的连续过程。近年来研究发现,表观遗传学的改变在肝癌的发生、发展过程中扮演着重要的角色。表观遗传的现象很多,已知的有DNA甲基化(DNAmethylation),基因组印记(genomic impriting),母体效应(maternal effects),基因沉默(gene silencing),核仁显性,休眠转座子激活和RNA编辑(RNA editing)等。其中抑癌基因RUNX3启动子区域的CpG岛被甲基化,导致该基因的表达沉默,成为近些年来研究肝癌相关发生发展机制及治疗的热点之一。研究报道,肝癌细胞中抑癌基因RUNT相关转录因子3(Runt-related transcription factor3, RUNX3)由于CpG岛启动子高甲基化,其在mRNA水平及蛋白水平表达明显下调。5-氮杂-2’-脱氧胞嘧啶核苷(5-Aza-2’-deoxycytidine,5-Aza-dC)是一种核苷酸类甲基转移酶抑制剂,也可以对DNA甲基转移酶进行特异性抑制,使DNA甲基化程度下降,进而逆转因甲基化而沉默的基因重新开始表达。在肝癌组织中RUNX3基因CpG岛甲基化的相关研究已有不少报道,而5-Aza-dC在肝癌细胞系SMMC-7721中对该基因甲基化状态影响的研究尚不多见。目的探讨去甲基化制剂5-Aza-dC对肝癌细胞株SMMC-7721抑癌基因RUNX3甲基化状态、mRNA表达水平及肝癌细胞生长周期的影响。材料与方法肝癌细胞SMMC-7721由河南省肿瘤医院中心实验室所赠,选择在RPMI-1640培养液(10%胎牛血清、100U/ml青霉素、100mg/ml)中培养,培养液置于37℃,饱和湿度的细胞培养箱中。每2到3天换液传代1次。并分为对照组(不进行加药处理)和5-Aza-dC处理组。1不同浓度的5-Aza-dC对肝癌细胞SMMC-7721RUNX3基因启动子区域甲基化状态的影响：分别以O.5μmol/L、1.5μmol/L、4.5μmol/L、13.5μmol/L、浓度的5-Aza-dC培养肝癌细胞SMMC-772172h,提取细胞DNA,参照甲基化特异性聚合酶链式反应(methylation-specific polymerase chain reaction,MSP)技术,分别设计甲基化和非甲基化引物,检测两组肝癌细胞RUNX3基因启动子区域的甲基化状态。2不同浓度的5-Aza-dC对肝癌细胞SMMC-7721RUNX3基因mRNA的作用：分别以0.5μmol/L、1.5μmol/L、4.5μmol/L、13.5μmol/L浓度的5-Aza-dC培养肝癌细胞SMMC-772172h,提取细胞RNA,应用RT-PCR(Real-time PCR)技术检测两组肝癌细胞SMMC-7721中RUNX3mRNA表达的影响。3不同浓度的5-Aza-dC对肝癌细胞SMMC-7721细胞周期的影响：分别以0.5μmol/L、1.5μmol/L、4.5μmol/L、13.5μmol/L浓度的5-Aza-dC培养肝癌细胞SMMC-772172h,收集细胞,使用流式细胞术(FCM)检测两组肝癌细胞SMMC-7721细胞周期的影响。4统计学处理：采用SPSS18.0统计软件对数据进行分析,结果以均数±标准差(x±s)表示,以p<0.05为差异有统计学意义。结果1不同浓度的5-Aza-dC对肝癌细胞SMMC-7721RUNX3基因启动子区域甲基化状态的影响：MSP结果显示,对照组肝癌细胞SMMC-7721RUNX3基因启动子区域存在高甲基化。在分别以0.5μmol/L、1.5μmol/L、4.5μmol/L、13.5μmol/L浓度的5-Aza-dC培养肝癌细胞SMMC-772172h后,肝癌细胞SMMC-7721RUNX3基因启动子区域甲基化状态随药物浓度增加而出现不同程度的逆转,逆转程度呈现出药物浓度依赖性的趋势；25-Aza-dC对肝癌细胞SMMC-7721RUNX3基因mRNA的影响：RT-PCR检测结果发现,对照组肝癌细胞SMMC-7721RUNX3mRNA未能检测到表达,经过O.5μmol/L、1.5μmol/L、4.5μmol/L、13.5μmol/L浓度的5-Aza-dC作用72h后,RUNX3的mRNA相对表达量分别为0.33±0.04、0.94±0.08、1.23±0.05和1.90±0.03,RUNX3的mRNA水平出现再表达,并呈现出浓度依赖方式增加。双变量相关分析说明RUNX3mRNA水平与5-Aza-dC的浓度呈显著正相关(r>0.95,P<0.05),差异有统计学意义。35-Aza-dC对肝癌细胞SMMC-7721生长周期的影响：FCM结果显示,不同浓度5-Aza-dC作用72h后,肝癌细胞SMMC-7721S期细胞比例随药物浓度增加而增加,提示5-Aza-dC可以诱导肝癌细胞SMMC-7721阻滞于S期。S期细胞比率与5-Aza-dC的浓度呈显著性正相关(r>0.95,P<0.02)。结论肝癌细胞SMMC-7721抑癌基因RUNX3启动子区域处于高甲基化状态。5-Aza-dC可逆转肝癌细胞SMMC-7721抑癌基因RUNX3启动子区域的甲基化状态,使得因甲基化而失活的RUNX3基因再表达,发挥抑癌作用,并可诱导肝癌细胞株SMMC-7721阻滞于S期,该作用随药物浓度增加而增强,从而抑制肿瘤生长。更多还原

【Abstract】 BackgroundHepatocellular carcinoma is one of the common malignant tumors in our country and one of the main types of primary hepatocarcinoma. However, its pathogenesis has not been completely elucidated, and progression of hepatocellular carcinoma is a process that involves multiple genetic changes, multi-factor, multi-step process. Recent studies have found that epigenetic alterations in hepatocellular carcinogenesis, development process plays an important role. Epigenetic phenomena was known including DNA methylation, genomic imprinting, maternal effects, gene silencing in nucleolar dominance, dormant transposons activation and RNA edit. The tumor suppressor gene RUNX3promoter region of the CpG island is methylated, leading to the gene silencing, which become a research focus of hepatocellular carcinoma related to the occurrence and development mechanism and treatment of in recent years.Research reported that RUNX3gene in hepatoarcinoma cell is obviously down-regulated in the mRNA level and protein level because of CpG Island promoter hypermethylation.5-Aza-2’-deoxycytidine(5-Aza-dC) is a nucleotide such methytransferase inhibitors,which is also a DNA methyltransferase specific inhibition, and it can lead to DNA methylation degree drop, then reversed the silencing genes to restart to express. Related studies in RUNX3gene CpG island methylation in hepatocarcinoma cell have been reported, while the study on5-Aza-dCacting upon the gene in methylation status of hepatocarcinoma cell line SMMC-7721is seldom.ObjectiveTo explore the effect of demethylation agents5-Aza-dC on the expression and the promoter region methylation status of the RUNX3gene, cell growth cycle and drug sensitivity in SMMC-7721cells.Materials and methodsSMMC-7721cells were given by the Central Laboratory of Henan Tumor Hospital and cultured in the RPMI-1640medium (10%fetal bovine serum,100U/ml penicillin,100mg/ml). Culture fluid was placed at37degrees, saturated humidity cell culture box.. Every2to3days for the fluid passage1times. And cells were divided into control group (no treatment with5-Aza-dC) and5-Aza-2’-deoxycytidine treatment group.1The effects of different concentration of5-Aza-2’-deoxycytidine on RUNX3gene the promoter region methylation status in human hepatocarcinoma cell SMMC-7721:Hepatocarcinoma cells SMMC-7721were cultured in respectively0.5μmol/L、1.5μmol/L、4.5μmol/L、13.5μmol/L5-Aza-2’-deoxycytidine culture liquid for72h and DNA was extracted.Then according to methylation specific PCR technology, methylated and non-methylated detection of primers were designed respectively, and methylation specific PCR (MSP) technique was performed to invest the effect of5-Aza-dCon methylation status of RUNX3gene promoter region in two groups of hepatocellular carcinoma cells.2The effects of different concentration of5-Aza-2’-deoxycytidine on RUNX3gene mRNA level in human hepatocarcinoma cell SMMC-7721:Hepatocarcinoma cells SMMC-7721were cultured in respectively0.5μmol/L、1.5μmol/L、4.5μmol/L、13.5μmol/L5-Aza-2’-deoxycytidine culture liquid for72h and RNA was extracted, RT-PCR(Real-time PCR) technique was used to detect the effect on expression of RUNX3mRNA in two groups of hepatocellular carcinoma cells..3The effects of different concentration of5-Aza-2’-deoxycytidine on cell cycle in human hepatocarcinoma cell SMMC-7721:Hepatocarcinoma cells SMMC-7721were cultured in respectively0.5μmol/L、1.5μmol/L、4.5μmol/L、13.5μmol/L5-Aza-2’-deoxycytidine culture liquid for72h and collect cells, the flow cytometry (FCM) technique was used to invested the effect on cell cycle in two groups of SMMC-7721hepatoma cells.4Data analysis:statistical software SPSS18.0was used for data analysis, data was showed as mean±Standard deviation(-x±s), p<0.05was considered that the difference was statistically significant.Results1The effects of different concentration of5-Aza-2’-deoxycytidine on RUNX3gene the promoter region methylation status in human hepatocarcinoma cells SMMC-7721:MSP results showed that RUNX3gene promoter region was at high methylation status in the control group of hepatocarcinoma cells SMMC-7721, and RUNX3gene promoter region methylation status was increasingly reversed with the concentration of5-Aza-dC gorwing,after hepatocellular carcinoma cells SMMC-7721were cultured in respectively0.5μmol/L.,1.5μmol/L、.4.5μmol/L、13.5μmol/L5-Aza-2’-deoxycytidine culture liquid for72h.2The effects of different concentration of5-Aza-2’-deoxycytidine on RUNX3gene the promoter region methylation status in human hepatocarcinoma cells SMMC-7721:Real-time PCR(RT-PCR) detection results showed that RUNX3mRNA level was not detected in control group of hepatocarcinoma cells SMMC-7721, after hepatocellular carcinoma cells SMMC-7721were cultured in respectively0.5μmol/L、1.5μmol/L、4.5μmol/L、13.5μmol/L5-Aza-2’-deoxycytidine culture liquid for72h.,RUNX3mRNA expression levels were0.33±0.04,0.94±0.08,1.23±0.05and1.90±0.03,RUNX3mRNA levels were increased in the control group.And results reflected a growth with concentration dependent manner.RUNX3mRNA level and 5-Aza-dC concentrations were positively correlated (r>0.95, P<0.05), the differences were statistically significant.3The effects of different concentration of5-Aza-2’-deoxycytidine on cell cycle in human hepatocarcinoma cell SMMC-7721:FCM results showed that after treatment of different concentrations of5-Aza-dC for72h,SMMC-7721hepatocarcinoma cell ratio in S phase increased with the drug concentration suggesting that5-Aza-Dc can induce SMMC-7721hepatocarcinoma cells arrested in S phase; S phase cell ratios increased in a concentration dependent manner. There was a a significant positive correlation between S phase cell ratio and the concentration of5-Aza-dC(r>0.95, P<0.05).ConclusionRUNX3gene promoter region was at high methyl ation status in hepatocarcinoma cells SMMC-7721.5-Aza-dC is able to reverse RUNX3gene promoter region methylation status in hepatic cancer cell SMMC-7721, upregulate RUNX3gene expression,and induce hepatoma cells to be arrested in S phase.And there was a significant positive correlation between the effect and the drug concentration.更多还原